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JinJun Zhang, Ali Hussain, John Marshall; Laser Mediated Activation Of Human Retinal Pigment Epithelial (rpe) Cells And Concomitant Release Of Matrix Metalloproteinases (mmps). Invest. Ophthalmol. Vis. Sci. 2012;53(14):5817.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate cellular dynamics and associated matrix metalloproteinase (MMP) release patterns of human retinal pigment epithelium (RPE) cells subsequent to irradiation by nanosecond pulsed laser at energy levels below visual threshold.
Human RPE-Bruch’s-Choroid (RPE-BC) explants were maintained under conditions of organ culture for 3 days and baseline MMP release patterns established. The explants were then irradiated with a nanosecond laser system (Q-switched, frequency doubled YAG laser, 532nm) using a 400μm spot size with a discontinuous energy distribution and total irradiance of 240mJ/cm2, and returned to the incubator for a further 14 days. At selected intervals, confocal laser scanning, conventional microscopy, cell viability, and proliferation assays were used to assess RPE cellular dynamics. Following laser treatment, media samples were obtained every 24 hours for quantification of MMPs 2&9 by standard gelatine zymography and densitometry.
Within four hours of laser intervention, 47 ±8% (Mean ± S.E.M, n=6) of the RPE cells within the treatment zone showed clear signs of injury. Most of the damaged RPE was characterised by some degree of cell membrane disruption with some cells showing structural disintegration. By post-treatment days 10-14, most of the injured beds were re-populated by migrating RPE cells from regions surrounding the lesion. The laser treatment was associated with differential effects on the components of the MMP system. Release of inactive MMP-2 (pro-enzyme) was little altered over the two-week experimental period whereas levels of inactive MMP-9 (pro-enzyme) increased 1.3-fold by day 1 to reach a 2.8-fold threshold by day 7 (n=4; p<0.005). However, changes in activated MMPs 2 & 9 were much more profound with levels increasing 6.7±2.6 fold (mean± S.E.M, n=4; p<0.005) and 4.4±1.1 fold (mean± S.E.M, n=4; p<0.05) respectively above controls at day 7 post laser.
The nanosecond laser with a discontinuous modal energy distribution producing non-visible lesions resulted in diffuse loss of RPE cells followed by re-population by cells migrating from the periphery of the lesions. The migratory response was accompanied by a robust but transitory release of activated matrix metalloproteinase enzymes. The collateral effect of this enzymic release on the structural aspects of underlying Bruch’s may be beneficial for addressing the patho-physiological features associated with both normal and advanced ageing of the membrane.
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